Subclinical leaflet thrombus in patients with severe aortic stenosis and atrial fibrillation -ENRICH-AF TAVI study

Subclinical leaflet thrombosis (SLT) can be one of the causes of transcatheter heart valve (THV) failure after transcatheter aortic valve implantation (TAVI). We sought to clarify the formation process of SLT and thrombogenicity during the perioperative period of TAVI. This multicenter, prospective, single-arm interventional study enrolled 26 patients treated with edoxaban for atrial fibrillation and who underwent TAVI for severe aortic stenosis between September 2018 and September 2022. We investigated changes in maximal leaflet thickness detected by contrast-enhanced computed tomography between 1 week and 3 months after TAVI in 18 patients and measured the thrombogenicity by Total Thrombus-formation Analysis System (T-TAS) and flow stagnation volume by computational fluid dynamics (CFD) (n = 11). SLT was observed in 16.7% (3/18) at 1 week, but decreased to 5.9% (1/17) at 3 months after TAVI. Patients with SLT at 1 week had a significantly decreased maximal leaflet thickness compared to those without SLT. Thrombogenicity assessed by T-TAS decreased markedly at 1 week and tended to increase at 3 months. The stagnation volume assessed by CFD was positively associated with a higher maximum leaflet thickness. This study showed the course of leaflet thrombus formation and visualization of stagnation in neo-sinus of THV in the acute phase after TAVI.


1.Assessment of vWF-HMW multimer
To assess the change of vWF-HMW multimer during peri-procedural period, we performed vWF multimer analysis.A plasma sample from each patient was used for vWF multimer analysis (based on the established method of sodium dodecyl sulfate agarose gel electrophoresis 1 ) outsourced to SRL, Inc. (Tokyo, Japan).The value of vWF-HMW multimers was estimated using Image J software and represented the relative amount of the largest multimers in the sample compared with those of the normal pooled plasma in the next lane of the same gel, and expressed as vWF-HMW multimer index (%).This index is defined as the ratio of the patients' vWF-HMW multimer ratio to the healthy control's ratio, as described previously 2,3 .

2.Measurement of plasma concentration of edoxaban
Trough concentration of edoxaban at 1 week after TAVI was quantified by using the commercially available standardized assay kit (BIOPHEN DiXaI kit, HYPHEN BioMed, Paris, France), which is an anti-Xa chromogenic method for the in vitro quantitative determination of direct Factor Xa inhibitors in human plasma, according to the protocol supplied by the manufacturer.

3.Stagnation volume assessed by CFD analysis
CFD analysis can visualize blood flow condition and can quantify the flow streamline, flow velocity, WSS, oscillatory index, and other parameters, using a patient-specific 3D model based on the individual CT images.Using contrast-enhanced CT at 1 week post-TAVI, CFD analysis for measuring stagnation volume was conducted by Cardio Flow Design, Inc. (Tokyo, Japan), according to the method established previously 4,5,6,7 .Briefly, 3-dimensional patient-specific geometries of the area extending from the aortic root to the ascending aorta was reconstructed from the individual CT images.Computational

4.Sample size calculation
The primary endpoint of this study was the difference in maximal leaflet thickness between 1 week and 3 months after TAVI.For sample size calculation, the number of cases that can verify the difference in maximum leaflet thickness between 1 week and 3 months after TAVI should be registered.However, since there is no information regarding the maximum leaflet thickness in patients treated with edoxaban, the calculation should be based on the maximum leaflet thickness 1 week and 3 months after TAVI in patients treated with vitamin K antagonist phenprocoumon.Previous study showed that rate of patients with leaflet thickening in at least one valve leaflet was 9.7% in contrast-enhanced CT 5 days after TAVI, and the maximum leaflet thickness was 3.27mm at median 5 days and 0.7mm at 3 months after TAVI, with a standard deviation of 1.5mm for the difference 10 .
Therefore, we set the following hypotheses: 1) the percentage of patients treated with edoxaban who exhibit valve leaflet thickening 1 week after TAVI was 9.7%.2) the maximum leaflet thickness 1 week after TAVI in patients treated with edoxaban was 3.27mm.3) The maximum leaflet thickness 3 months after TAVI in patients treated with edoxaban was decreased to 0.7mm.4) the standard deviation of the difference between 1 week and 3 months after TAVI in patients treated with edoxaban was 1.5mm.At a power of 90% and a significance level of 5%, the required number of cases to verify the effect was 83 cases.Assuming a dropout rate of 15%, 98 cases were calculated as the required sample size for enrollment.However, our study faced recruitment difficulties due to the COVID-19 pandemic.We could not reach the target sample size of 98 patients, and we terminated the study with 26 patients.

Supplementary
meshes were generated by the ANSYS-ICEM 16.0 software (ANSYS Inc., Tokyo), and the finite volume solver package ANSYS Fluent 18.0 (ANSYS Inc.) was used to solve the Navier-Stokes equation of incompressible transient Newtonian fluid in setting.The boundary condition was set as follows; mean aortic root inflow rate of 4,450mL/min, mean left coronary artery flow rate of 148 mL/min, mean right coronary artery flow rate of 74.2mL/min, and aortic root outflow pressure of 96.0 mmHg.The pulsatile flow was employed, and the stagnation volume of the aortic root with THV was calculated separately during systole and diastole 8,9 .Fluid-Structure Interaction analysis was not performed because it was not possible to calculate the movements of valves and blood vessel walls.Stagnation was defined as velocity <0.01 m/s at systole or velocity <0.001 m/s at diastole.The mean stagnation volume for the comparison of blood flow stagnation was calculated, as shown in the Supplementary Figure 2.

Table 1 . Baseline characteristics of study population
AF indicates atrial fibrillation; CAD, coronary artery disease; ACE, angiotensin converting enzyme; ARB, angiotensin II receptor blocker; ARNI, angiotensin receptor neprilysin inhibitor; SGLT, sodium glucose transporter; pressure gradient; AR-AUC, area under the curve for the atheroma chip; PL-AUC, area under the curve for the platelet chip.